Rana pipiens pipiens, the northern leopard (grass) frog, along with at least three other species or subspecies form the Rana pipiens complex. The nation wide distribution and availability of the members of the complex have contributed significantly to the popularity of R. pipiens as an experimental organism in biomedical research. There is increasing evidence of significant physiological differences among members of the complex. Schmid (Chromosoma, 1978) published an analysis of the C-banding patterns of the southern variety of the complex. Our own investigations on the C-banding patterns of the northern variety indicate differences in the karyotypes of the two varieties. The specific aims of this project are to develop "banded signatures," C, G and Q-bands (the latter two techniques are to be developed) for each chromosome in the karyotype of different geographic varieties of R. pipiens. This will provide a cellular mechanism for differentiating between members of the complex. This base line information will provide a broad basis on which to analyze chromosomal relationships between members of the complex and their hybrids. It will also provide a mechanism for analyzing genomic changes in established cell lines and derivatives of these lines. It may also provide information on the physiological differences betgween members of the complex at the whole animal, cellular and subcellular level, i.e., incompatibilities between nuclei and cytoplasms of certain geographic varieties. Metaphase chromosome spreads will be prepared from leukocyte cultures and C-banded by treatment with alkali. The banding patterns of the geographic varieties will be compared. Current G and Q-banding techniques will be modified to accommodate them to amphibian chromosomes in order to provide a more detailed view of the respective karyotypes. The establishment of "banded signatures" for individual chromosomes from the geographic varieties will allow the specific identification of individual chromosomes. This information will be useful in analyzing phylogenetic relationships and chromosomal variations in R. pipiens tissues and cells employed in biomedical research.